: Errors in maintaining the physical integrity of the genome are the underlying cause of many cancer-prone genetic defects, and cells derived from many tumors exhibit aneuploidy and chromosomal aberrations. However, programmed DNA double-strand breaks (DSBs) also play a vital role in cellular development, as the initiator of homologous recombination during meiosis and the developmentally determined non-homologous rearrangement of the genes for B cell and T cell receptors. Nevertheless, despite these crucial roles in development, DNA double-strand breaks are among the most dangerous forms of DNA damage, because failure to repair them properly can lead to loss of genetic material, or to chromosomal rearrangements and profound alterations to gene expression. Thus all cells have evolved mechanisms for efficient double-strand break repair (DSBR), and many of the protein components have been conserved throughout eukaryotic evolution, demonstrating the critical role these mechanisms play in the life the organism. Although many of the proteins needed for this reaction have been identified, the molecular mechanisms have not been delineated. A limiting factor is the absence of easily manipulated systems for monitoring repair of DSBs created in vivo. An adenovirus vector system in which DSBs are created in target viral genomes containing the HO recognition site, using the yeast mating-type switch (HO) endonuclease genome, has been developed. The newly created DSBs are repaired by the DSBR machinery of the cell, but only if viral early region 4 ORF6 (E4orf6) expression is prevented. In this proposal, the adenovirus HO system will used: 1) to investigate the mechanism of DSBR in vivo; 2) to determine the basis of the inhibition of DSBR by E4orf6. The adenovirus HO vectors will be used to create completely non-complementary 3' DNA ends. Analysis of junctional sequences will be used to distinguish between the various models for non-homologous end-joining. The adenovirus vectors will also be used to determine the DSBR capacity of cells that are sensitive to ionizing radiation (IR). If possible, viral vectors expressing conventional restriction enzymes will be used to examine DSBR of different end structures, and to examine DSBR in the IR-sensitive cells. The second aim is to uncover the mechanism of inhibition of DSBR by E4orf6. E4orf6 is necessary but not sufficient for inhibition of BR. Experiments are designed to test the hypothesis that one of the viral partners is the ElB 55kDa protein, which interacts physically and functionally with E4orf6. E4orf6 also interacts with two specific cellular proteins, p53 and DNA-PK. Experiments are proposed to test the importance of these interactions to the inhibition of DSBR. Biochemical analysis of the inhibition of DSBR by E4orf6 will use an in vitro system for the assaying DSBR, developed previously. Nuclear extracts will be made from cells infected with the appropriate viral vectors to see if DSBR is inhibited by E4orf6 and its necessary partner proteins. In addition, the kinase activity of DNA-PK will be measured to see if it is inhibited by E4orf6.